1. Field of the Invention
The present invention relates to the use of Flp recombinase to catalyze FRT site-specific DNA recombination in a transgenic non-human mammal, preferably a mouse.
2. Description of the Related Art
Site-specific recombinases are being developed as tools for genetic engineering because of their simplicity and precise activity in a variety of organisms. Two well studied recombinases are Flp and Cre. For use in vivo, a recombinase should be active in a transgenic non-human mammal. While Cre-mediated recombination has been successfully employed in trangenic mice, the utility of Flp recombinase in transgenic mice has not previously been established.
U.S. Pat. No. 4,959,317 discloses the use of the Cre-lox recombinase system in yeast and cultured mammalian cells, but not in transgenic mice. Other site-specific recombinases were not discussed.
U.S. Pat. No. 5,527,695 demonstrates the use of Flp recombinase in plants, but not in cultured mammalian cells or transgenic mice. A number of different site-specific recombinase systems are discussed; however, no guidance appears to be given for selecting among the different systems and their use in a transgenic mouse is not discussed.
Kilby et al. (1993) reviewed the demonstrated activities of different site-specific recombinases in cells and organisms. Table 1 shows that, to their knowledge, Flp recombinase activity in transgenic mice had not been accomplished.
Flp-mediated deletion was demonstrated in embryonic stem (ES) cells by Jung et al. (1993). Gu et al. (1993) compared the activity of Cre and Flp recombinases in ES cells and found xe2x80x9ca major fraction of ES cells transiently transfected by the cre vector undergo Cre-loxP-mediated gene deletion (which is not the case in our hands if the related FLP/FRT system from yeast is used [Jung et al., 1993, and unpublished data])xe2x80x9d. Both papers were contributed by the Rajewsky group, and the same group has exclusively used the Cre-loxP system in transgenic mice to inactivate endogenous genes, instead of Flp recombinase (Gu et al., 1994; Kxc3xchn et al., 1995). Thus, prior to the present invention, it was thought that using Cre recombinase was preferred over using Flp recombinase.
In view of the above teachings of the related art, it is an unexpected finding of the present invention that Flp recombinase can function in a developing mammal to catalyze FRT site-specific recombination.
Moreover, although Cre recombinase has been successfully used to create specific deletions in the mouse genome, the general utility of Cre to catalyze recombination is currently being established. Therefore, an additional method is needed for generating site-specific genetic alterations in the following ways: (1) a site-specific recombinase demonstrating a different dose-sensitivity could be used in situations where proper regulation of the recombination event cannot be achieved using Cre and (2) two site-specific recombinases could be used in vivo to engineer simultaneous or sequential recombination reactions (e.g., independent gene activation or inactivation events).
For example, site-specific recombinases may be used to activate expression of a tracer molecule to mark cell lineages. Factors that influence the determination of these cell lineages can be identified by analyzing these marked cells in the genetic background of various mutations, including mutations generated using the second recombinase system. Additionally, having access to two recombinase systems allows for efficient use of the first recombinase to generate a mutation, and the second recombinase to remove any selectable markers used in generating that mutation which, if left in place, would confound interpretation of the study. A second recombinase system is desired which exploits the ability of Flp recombinase to catalyze FRT-specific recombination in a transgenic non-human mammal which can be used alone or to expand the uses of the CrelloxP system.
The present invention provides a transgenic non-human mammal with sufficient Flp recombinase activity to catalyze recombination between FRT sequences, a transgenic non-human mammal containing FRT target nucleic acid which serves as an efficient substrate for Flp, a process of in vivo gene manipulation using the transgenic non-human mammals, and a genetic system comprised of the Flp transgenic non-human mammal and the FRT target transgenic non-human mammal which contains at least one FRT sequence.
An object of the invention is to provide a transgenic non-human mammal with Flp recombinase activity useful for manipulation of the genome in the intact mammal.
Yet another object of the invention is to provide a process of in vivo genetic engineering using Flp recombinase activity to catalyze FRT site-specific recombination in a non-human mammal.
A further object of the invention is to provide a genetic system of the transgenic non-human mammal with Flp recombinase activity and at least one nucleic acid which is a substrate for Flp recombinase (e.g., the nucleic acid contains a FRT sequence). The nucleic acid may also contain a transgene for insertion into the genome of a non-human mammal, or a region which directs homologous recombination into the genome of a non-human mammal.
In one embodiment of the invention, a transgenic non-human mammal is provided which contains a Flp transgene integrated in its genome. Optionally, at least one Flp-recognition sequence is also integrated in the genome of the transgenic non-human mammal. The Flp-recognition sequence comprises FRT or a derivative thereof such as, for example, SEQ ID NO:14 or SEQ ID NO:15. A transgenic non-human mammal of the invention contains sufficient Flp recombinase activity in a cell to catalyze recombination between Flp-recognition sequences of the cell, chromosomal and/or extrachromosomal. Flp recombinase activity may be regulated by a chemical (e.g., exogenously administered drug, endogenous metabolite), the mammal""s developmental stage, its body temperature, or tissue type of the cell.
The substrate for Flp recombinase activity is a Flp-recognition sequence. The genome of the transgenic non-human mammal may comprise one Flp-recognition sequence, two Flp-recognition sequences, or more than two Flp-recognition sequences. A chimeric or mosaic transgenic non-human mammal may contain cells with different numbers of Flp-recognition sequences due to Flp-mediated recombination; when a Flp-recognition sequence is integrated on only one of the pair of homologous chromosomes, the genome will be hemizygous for the Flp-recognition sequence.
Recombination between two Flp-recognition sequences integrated on different chromosomes results in translocation between those chromosomes. Such translocations are a common means of creating mutations that lead to developmental abnormalities or tumorigenesis.
Recombination between two Flp-recognition sequences in direct repeat orientation may cause excision of an intervening DNA sequence (e.g., a gene). Although such events are potentially reversible because Flp-mediated recombination is conservative, loss of the excised DNA sequence during cell division or by degradation makes the mutation irreversible. A null mutation in any gene may be created in this way, and the function of the gene studied in specific cells and/or at specific developmental stages.
Recombination between two Flp-recognition sequences in inverted repeat orientation may cause inversion of an intervening sequence or gene. As in Salmonella phase variation, inversion may cause activation or inactivation of a gene. If gene activity is detectable (e.g., selectable marker, histochemical marker, reporter gene), cell lineages may be traced by identifying recombination events that mark a cell and its descendants through detection of gene activation or inactivation. Cell lineages may be traced independent of gene activity, by monitoring differences in the integration site of the Flp substrate.
Recombination between a Flp-recognition sequence integrated on a chromosome and a Flp-recognition sequence integrated on extrachromosomal genetic material may cause insertion of the genetic material into the chromosome. An insertion created in this manner would provide means for creating transgenic non-human mammals with site-specific integration of a single copy of the transgene at a site in the genome specified by the chromosomal Flp-recognition sequence. Transgene insertion at a defined site in the genome would ensure reproducibility of expression because confounding effects of variable chromatin structure would be minimized.
Preferably, the intervening sequence or genetic material contains a gene such as, for example, a developmental gene, essential gene, cytokine gene, neurotransmitter gene, neurotransmitter receptor gene, oncogene, tumor suppressor gene, selectable marker, or histochemical marker, or portion thereof. Recombination may cause activation or inactivation of a gene by juxtaposition of regulatory regions to the gene or separation of regulatory regions from the gene, respectively.
The transgenic non-human mammal of the invention may also contain a Cre recombinase transgene. A cell of the transgenic non-human mammal would contain sufficient Cre recombinase activity to catalyze recombination between Cre-recognition sequences (e.g., lox site) of the cell.
A second embodiment of the invention is a process for in vivo genetic engineering using the transgenic non-human mammal. Flp recombinase activity is induced in a cell containing at least two Flp-recognition sequences at a level sufficient to catalyze site-specific recombination in the cell. This results in recombination between Flp-recognition sequences in the cell. The cell may be of germ line or somatic origin. If recombination occurs in a germ cell or a totipotent cell, offspring may be produced with a genome altered by the recombination event. A process for studying carcinogenesis and its treatment is provided by using Flp-mediated recombination to cause activation of an oncogene or inactivation of a tumor suppressor gene. Candidate compounds or compositions may be screened in such a process to identify candidates that act to promote carcinogenesis (i.e., a cancer promoter) or inhibit carcinogenesis (i.e., a cancer inhibitor). Similarly, Flp-mediated recombination in a transgenic non-human mammal may be used in a process of activating ectopic expression of a gene during development, inactivating expression of a gene at a specific time or in a specific tissue, or identifying a cell lineage by activation or inactivation of a gene. The gene may be a developmental gene, essential gene, cytokine gene, neurotransmitter gene, neurotransmitter receptor gene, oncogene, tumor suppressor gene, selectable marker, or histochemical marker.
In a third embodiment of the invention, a genetic system comprising the transgenic non-human mammal and a purified nucleic acid containing at least one Flp-recognition sequence is provided. Preferred nucleic acids include the vectors described herein. Optionally, the genetic system may also comprise means for producing a transgenic non-human mammal which contains at least some portion of the purified nucleic acid; preferably, at least the Flp-recognition sequence is integrated into the genome of the transgenic non-human mammal.
As used herein, a transgenic non-human mammal is a non-human mammal into which genetic material has been introduced with a recombinant nucleic acid. The introduced genetic material may become integrated into the genome of the non-human mammal, preferably stably- or excisably-integrated into a chromosome of the non-human mammal, and be transmitted through the germ line to a succeeding generation. Alternatively, the genetic material may be maintained as an episome or an artificial chromosome.
Any such introduced genetic material is termed a transgene. If the transgene is unstable or is excised during cell division, the result will be a mosaic mammal comprised of at least two cell types with different genetic content but derived from the same zygote. Such an mammal may be used in cell lineage tracing; thus, a transgene flanked by FRT sites may be excised during ontogeny by the action of Flp recombinase. In contrast, a chimeric mammal comprises at least two genetically different cell types derived from different zygotes (e.g., a mammal resulting from injection of embryonal carcinoma or embryonic stem cells into a genetically different blastocyst). If the transgenic non-human mammal only contains the transgene in somatic cells, the transgene will not be passed to a succeeding generation through the germ line.
Preferably, the non-human mammal is a mammal for which methods to introduce a transgene are known in the art such as, for example, cow, goat, mouse, pig, rabbit, rat and sheep. Such methods of introducing genetic material include microinjection for the creation of transgenics from zygotes; and electroporation, biolistics, lipofection, calcium phosphate-DNA co-precipitation, DEAE-dextran, microinjection, and viral infection for the creation of transgenics from a cultured cell (e.g., pluripotent cells such as a teratocarcinoma or embryonal carcinoma, totipotent cells such as an embryonic stem cell) and subsequent transfer into an embryo.
The transgenic non-human mammal, the process, and the genetic system are particularly advantageous when separate control of more than one recombination event is desired in the mammalian genome. For example, integration of a loxP sequence-containing substrate may be followed by deletion of a selectable marker from the substrate which is mediated by Flp recombinase activity catalyzing recombination between FRT sequences flanking the selectable marker, and Cre-mediated recombination of the loxP-containing substrate. Integration of the loxP sequence containing substrate is preferably by homologous recombination, and expression of the co-integrated selectable marker enriches for this rare event; the second event occurs by Flp-mediated site-specific recombination which deletes the selectable marker to reduce competition from the regulatory regions of the selectable marker; and the third event of deleting gene sequences lying between loxP sites occurs by Cre-mediated site-specific recombination.